siRNA molecules are short double-stranded RNA duplexes; each strand has between 19 and 25 bases. The prototypical siRNA molecule used most often is 21 bases, with a 19 base pair duplex region and 2 base 3’ overhangs consisting of thymidine deoxyribonucleotide residues.

These molecules have been used extensively in RNAi research, but recent developments show these molecules can have some undesired effects:

Eliciting a host interferon response — It was thought that RNA duplexes of less than 30 nucleotides did not elicit an interferon response. However, researchers have found many instances where standard siRNA stimulates host immune responses, particularly in certain cell types or if specific sequence motifs are present. This non-specific immune response to dsRNA can render experimental data useless.

Generating off-target knockdown effects — Because both the sense and anti-sense strands of the duplex can participate in the RNAi pathway, the sense strand of the siRNA molecule may elicit an off-target RNAi knockdown effect by binding to an unintended mRNA. And if either siRNA strand has partial homology to an off-target mRNA (one to several mismatches) this can elicit a “miRNA-like” effect, resulting in a milder knockdown event and confusing data interpretation.

Short half life — Perhaps the most important limitation of standard siRNA molecules for in vivo experiments. Fortunately, advances in RNA modification technologies have resulted in RNA duplexes with greater specificity, reduced risk of stimulating an interferon response, and increased stability.

Stealth RNAi™ siRNA

Stealth RNAi™ siRNA, a chemically modified 25-mer blunt-ended RNA duplex, is an ideal siRNA molecule for performing in vivo studies. The Stealth RNAi™ siRNA duplex has been chemically altered so only the antisense strand participates in the RNAi pathway, greatly decreasing the potential for off-target effects. Additionally, the chemical modification also allows Stealth RNAi™ siRNA to avoid stimulating a host immune response.

Using Stealth RNAi™ siRNA for in vivo experiments greatly increased half-life both in vitro and in vivo, as compared to standard siRNA. This added stability is extremely important for in vivo experiments, given the nuclease-rich environment within an organism. All of these factors combined make Stealth RNAi™ siRNA the ideal synthetic RNAi molecule for in vivo experiments.

Special considerations for synthetic molecules for in vivo RNAi experiment

When performing in vivo RNAi experiments with synthetic RNA duplexes, it is very important to have a well-defined, non-toxic, and sterile starting material, compatible with physiological conditions.

Invitrogen now offers an “in vivo” purity for both BLOCK-iT™ siRNA and Stealth RNAi™ siRNA for in vivo RNAi experiments. This “in vivo” purity has several components:

After standard synthesis using high-quality starting materials, RNA oligos are duplexed and desalted.

At this point, the researcher can also request HPLC purification to enrich for full-length duplexes, but this step is not necessary for in vivo RNAi.

Then, duplexes are put through a series of dialysis and counter-ion exchange steps which replace potentially toxic ammonium counter-ions with sodium, and also lower the conductivity to physiological conditions.

Finally, duplexes are sterile filtered and lyophilized for shipment.

Endotoxin testing of the final product is also available as an additional option.

These RNA duplexes are available in very small (for in vitro validation) up to very large quantities (for large-scale animal studies). Additionally, these “in vivo” purity RNA duplexes can be ordered with a variety of modifications, including phosphate, biotin, and Alexa Fluor® dyes and can be ordered using our simple BLOCK-iT™ RNAi Express for in vivo synthetics.